The field of regenerative medicine has focused on the use of biochemical factors, such as cytokines and morphogens, to modulate stem cell behavior and tissue outcomes. In contrast, endogenous bioelectric signals (steady-state resting potential or Vmem) have long been identified as a key factor in tissue development and tissue repair, but have only received very limited attention towards the field of regenerative medicine. In our current grant we have made major progress in elucidating the role of Vmem in modulating stem cell behavior and tissue repair as a transformative application for regenerative medicine. In this renewal application, we plan to gain further mechanistic insight into signaling by changes of Vmem in non-neural cells as well as into the self-organizing properties of Vmem-regulatory networks to embed desired Vmem patterns into engineered tissues to direct tissue function. Our goal is to advance our understanding and implementation of bioelectric signaling as a tool for regenerative medicine applications. Our hypothesis is that a better understanding of bioelectric signaling on the molecular and cellular levels has the potential to impact tissue engineering, stem cell therapies, and tissue regeneration. In our two proposed aims we will: (Aim 1) dig deeper into the mechanisms governing how voltage signals are transduced into cellular responses, and (Aim 2) we will explore how to incorporate bioelectric patterning in 2D and 3D tissue models by specifying Vmem with high temporal and spatial control using optogenetics tools and patterned drug delivery. Through these Aims, we will address our hypothesis from two angles: the mechanistic, molecular basis of voltage signaling, and the broad, tissue-wide patterning application for which voltage signaling may be utilized. As in the current grant, this i a team approach with two PIs with complementary and synergistic skills, David Kaplan (Biomedical Engineering) with a focus on stem cells and tissue formation in vitro, and Michael Levin (Biology) with a focus on molecular tools for Vmem control and tissue development. The team has made substantive progress in the first 3-plus years on the current grant. Major impact to the field of regenerative medicine is anticipated with the plans proposed in this renewal program by harnessing bioelectric signaling. !